Our aim is to explore regulatory mechanisms involved in cell differentiation and malignant transformation in melanoma cell culture. These cells form tumors in all syngeneic mice injected; but are reversibly suppressed in malignancy, tyrosinase activity, and plasminogen activator (PA) upon growth with 1-3 micron g/ml of BrdU. Continuous growth with BrdU maintains the suppression, and cells so treated can immunize mice against the malignant parental line. Immunization is being studied both in vivo and in vitro. The kinetics of suppression of these multiple effects are being studied after 24, 48, and 72 hrs of growth (i.e., 1, 2, or 3 cell divisions) in BrdU- containing medium, as are the kinetics of return to the cells' pre- treatment state after growth in BrdU-free medium (RM) for up to 1 week. Bromouracil replaces about 23% of thymine residues after 24 hr, 40% after 48 hr, and 45% after 72 hr. Upon subsequent growth in RM extent of replacement declines in a manner consistent with dilution by new DNA synthesis, reaching 5-10% substitution by day 7. Tumorigenicity and expression of PA appear to be affected immediately by these changes; there is a lag in the time course of reduction and subsequent regaining of tyrosinase activity. Substitution by bromouracil in either strand of the DNA seems to be sufficient for suppression of these functions. We are planning to continue these studies using synchronized cells to aid in our probe of the mechanism of regulation of these functions. Bibliographic references: Silagi, S., and Wrathall, J.R. Reversible suppression of differentiation and malignancy in bromodeoxyuridine-treated melanoma cells. In: 4th Inter. Symp. of the Princess Takamatsu Cancer Research Fund, Univ. Tokyo Press, Tokyo, 369- 395 (1974). Christman, J.K., Silagi, S., Newcomb, E.W., Silverstein, S.C., and Acs, G. Correlated suppression by 5-bromodeoxyuridine of tumorigenicity and plasminogen activator in mouse melanoma cells. Proc. Nat. Acad Sci. USA, 72, 47-50 (1975).